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Interspecies variations in response to topical application of selected zinc compounds
Fd Chem. Toxic. Vol. 29, No. 1, pp. 57-64, 1991 Printed in Great Britain. All rights reserved
0278-6915/91 $3.00+ 0.00 Copyright© 1991PergamonPressple
INTERSPECIES VARIATIONS IN RESPONSE TO TOPICAL APPLICATION OF SELECTED ZINC COMPOUNDS A. B. G. LANSDOWN Chafing Cross and Westminster Medical School, Department of Comparative Biology, London W6 8RP, UK (Received 27 June 1990; revisions received 19 September 1990)
Abstract--The dermal irritancy of six zinc compounds was examined in three animal models. In open patch tests involving five daily applications, zinc chloride (1% aqueous solution) was severely irritant in rabbit, guinea-pig and mouse tests, inducing epidermal hyperplasia and ulceration; aqueous zinc acetate (20%) was slightly less irritant. Zinc oxide (20% suspension dilute Tween 80), zinc sulphate (1% aqueous solution) and zinc pyrithione (20% suspension) were not overtly irritant, but induced a marginal epidermal hyperplasia and increased hair growth. Zinc undeeylenate (20% suspension) was not irritant. Epidermal irritancy in these studies is related to the interaction of zinc ion with epidermal keratin. The compounds studied were not consistently baeteriostatic in the three species tested.
INTRODUCTION
the action of zinc in epidermal repair following incisional lesion, varicose ulcer or other forms of benign injury. Considerable clinical interest and commercial value has been attached to the premise that zinc accelerates the healing of skin wounds (Hallbook and Lanner, 1972; Hallmans, 1978; Henzel et al., 1970; Hussain, 1969; Pories et al., 1967), but the subject is controversial and contradictory evidence is available (Barcia, 1970; Groundwater and MacLeod, 1970; Lancet, 1975). Clearly, if zinc is beneficial to wound healing in the skin, one would expect it to promote cell proliferation, migration and differentiation in regenerating tissues. This effect is still not established clinically or experimentally (Marks et al., 1985). A variety of preparations containing zinc is available to treat skin lesions, but in many cases their action on the epidermis is unclear. As a prelude to further studies to define more clearly the potential value of zinc in wound healing, the study presented here was designed to observe the influence of specific zinc compounds on the epidermis of three animal models commonly used in experimental dermatology. This study resembles an earlier investigation that compared the irritancy of several aluminium compounds (Lansdown, 1973 and 1974). Like aluminium, zinc salts of strong acids are astringent and corrosive to the skin at high concentrations. However, whereas aluminium has no obvious role in skin physiology, zinc is an essential trace metal. Therefore, it is of additional interest to examine the extent to which exaggerated levels of topical zinc enhance the physiological action of the element, or induce toxic changes.
Zinc is an essential nutrient and constituent of all biological systems (Raulin, 1869). After iron, it is the most abundant trace metal in the mammalian body. Although much is still unclear about the physiological role of zinc, it is now known to be a component of at least 40 enzyme systems, including alkaline phosphatase, carbonic anhydrase, dehydrogenases, carboxypeptidases and D N A / R N A polymerases (Keller and Mann, 1940; Kirchessner et al., 1976). Zinc concentrations tend to be higher in the skin, red blood cells and tissues that exhibit high proliferative activity (Schroeder et aL, 1967). In human skin, six times more zinc is found in the epidermis than in the dermis (Molokhia and Portnoy, 1969). The importance of zinc in the growth and maintenance of the functional integrity of the skin is unequivocal. The rare lethal condition acrodermatitis enteropathica, which results from a hereditary hypozincaemia, is well documented as are skin lesions induced experimentally in laboratory animals fed zinc-deficient diets (DeGryse et al., 1987; Ecker and Schroeter, 1978; Moynahan, 1974; Ohlen and Scott, 1986; Prasad, 1966; Strain et al., 1966; Tucker et al., 1976). Zinc-responsive dermatosis occurs naturally in certain strains of dog (Tucker and Salmon, 1955; Van Herck et al., 1989). These conditions show similar dermatological features namely hyperkeratosis, parakeratosis, hypopigmentation and alopecia. This suggests that despite obvious morphological differences in the skin between species, animal models may be used to provide beneficial information that can advance our understanding of the pathophysiological role of zinc in human skin. Zinc concentrations are normally maintained in the body by intestinal absorption, but experimental evidence has shown that low levels of zinc may be absorbed percutaneously in zinc-deficient animals to restore norrnozincaemia (Kapur et al., 1974; Keen and Hurley, 1977; Skog and Wahlberg, 1964; Yankell, 1969). This may have further implications in
MATERIALS AND METHODS
Animals Mice. Animals used were TO (outbred) and AG2 (inbred) strains bred under barrier-maintained specified pathogen-free conditions at the Chafing Cross and Westminster Medical School. Male mice
57
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A.B.G. L^NSDOWS
were 8-10 wk old, and housed in a conventional environment (12-hr light/dark cycle, 2 2 _ 2°C and 55% relative humidity) and fed a complete rodent diet (CRM, Biosure, Cambridge, UK) (zinc content 65 mg/kg, w/w) and deionized water ad lib. Rabbits. Animals were New Zealand white males weighing 2.5--4.0kg, which were reared at the Chafing Cross and Westminster Medical School. They were housed singly under conditions of a 12-hr light/dark cycle, at 18 _ 2°C and 45% relative humidity. Their diet consisted of R14 pellets (Biosure) (zinc content 74 mg/kg, w/w) and tap-water. Guinea-pigs. Male animals of the Dunkin-Hartley white strain (250-400 g) were supplied by HarlanPorcellus (Heathfield, Sussex, UK). They were fed an RGP diet (Biosure) (zinc content 65 mg/kg, w/w) and maintained in an environment similar to that of the mice.
Chemicals The zinc compounds were supplied by the Sigma Chemical Co. Ltd (Poole, Dorset, UK) and had a minimum purity of 98%. The concentrations used were those that had been shown to be non-irritant in preliminary acute studies in white (TO) mice (mice back skin was treated with 0.5 ml of the test compound at 0.1, 1.0, 10 or 20% and examined for erythema after 24 hr). The following preparations were applied to the animal skins: (i) 1% (w/v) zinc chloride solution in deionized water; (ii) 1% (w/v) zinc sulphate solution in deionized water; (iii) 20% (w/v) zinc acetate solution in deionized water; (iv) 20% (w/v) zinc oxide suspension in 0.1% Tween 80; (v) 20% (w/v) zinc undecylenate suspension in 0.1% Tween 80; and (vi) 20% (w/v) zinc pyrithione suspension in 0.1% Tween 80. Control areas of skin were treated with either deionized water or Tween 80, both of which are non-irritant in subacute skin tests in mice (Lansdown and Grasso, 1972). In each case, test solutions were prepared 24 hr before use and stored at 4°C. The pH of each preparation was measured immediately before application (Table 3). Experimental procedures Four experiments were set up to compare the irritancy of the six zinc compounds. These included two studies in rabbits (one under open patch test conditions, and one using occlusive conditions), and one each in guinea-pigs and mice. In each case, test sites (5 cm 2) were prepared in the mid-dorsal regions. The hair coat was clipped closely and the skin cleansed with 70% alcohol and allowed to dry before treatment with zinc compounds. In the initial rabbit study, 4 animals were acclimatized to conventional restraining boxes. Eight test sites were designated, four on each side of the mid-dorsal line. Zinc compounds (or controls) were applied daily (0.5 ml) at 09.00-09.30 hr on 5 successive days. The animals were restrained for 30 min to allow test sites to dry. In a second rabbit experiment, 4 rabbits were prepared as above, but the 0.5 ml of test compound was applied to the skin on a sterile gauze pad that was secured by a hypoallergenic adhesive tape (Draize
et al., 1944; Lansdown, 1978). The trunks of these animals were wrapped in rubberized fabric and impermeable dressing for 3 days. At this stage, dressings were removed and skin sites evaluated for irritancy. Two rabbits were killed at this time by using excess barbiturate anaesthetic. The remaining animals were redressed with freshly impregnated gauze pads and then re-examined and killed after a further 2 days. In the guinea-pig experiment, 8 animals were prepared by shaving the dorsal skin to provide three treatment sites (5 cm:). A total of three treatment sites was thus available for each of the test and control solutions/suspensions. The guinea-pigs were treated daily (09.00-09.30 hr) for 5 consecutive days. The animals were restrained until the test sites had dried. Open patch tests were conducted in mice in eight groups of 6 animals. Sites of 5 cm 2 were treated with 0.5 ml of the test or control agent for 5 consecutive days. Animals were gently anaesthetized with ether until the test sites had dried. Twenty-four hr after the fifth daily treatment, rabbits, guinea-pigs and mice were killed using excess barbiturate anaesthetic or cervical dislocation (mice). Representative samples of each test and control skin site were preserved in 10% phosphate buffered formalin for histology. Thin sections cut along the anterior-posterior axis were stained with haematoxylin and eosin, or with morin dye, which fluoresces blue-green in the presence of zinc ions and ultraviolet light. Epidermal cell kinetics The influence of the various zinc preparations on the mitotic behaviour of the dorsal skin epidermis was examined. The experiment was set up identically to the mouse experiment, except that groups of 5 A2G mice were used for each treatment regimen. Twenty-four hr after the final treatment, each mouse received a single ip injection of 0.1 mg vincristine (Oncovin, Lilly, Basingstoke, Hants., UK) in 0.25 ml of normal saline (Bullough and Laurence, 1966). The animals were killed by cervical dislocation after 4 hr and three strips of skin from the treatment sites were preserved in 10% formalin for histological examination. Sections were stained with haematoxylin and eosin, and arrested mitoses per 1000 cells were counted. The mitotic index was calculated as: total no. of arrested mitoses x 100 total no. of cells counted Where the mouse skins exhibited acanthotic changes, and where mitoses occurred above the stratum germinativum, mitotic counts only included cells in the striatum germinativum and in the first layer of the stratum spinosum. The mitotic index was calculated for each mouse. Microbiology In view of the observation that certain zinc compounds exhibit antibiotic properties (Imokawa et al., 1982; Snyder et al., 1977), fresh skin samples were obtained from the rabbits, guinea-pigs and mice at the end of the experimental period. They were routinely smeared across sterile blood agar plates, incubated at 37°C, and examined for bacterial
Interspecies responses to zinc compounds growths 24 hr later using standard criteria. Previous quality assurance studies have shown an absence of ectoparasites, mycoplasma and viral infections in animals bred and maintained in the animal facility of the Charing Cross and Westminster Medical School.
Statistics Where appropriate, statistical comparisons were made with Student's t-test. RESULTS
Open patch tests Zinc chloride was clearly the strongest irritant of the compounds tested in open patch tests in the three species (Table 1). From the first application, skin test sites became erythematous, and by the fifth day they showed ulcerative changes. Lower levels of erythema were seen following treatment with zinc acetate, sulphate, pyrithione or undecylenate. These compounds did not induce ulceration or scaling in the test period. Zinc oxide suspension, dilute Tween 80 and deionized water also did not show an obvious effect. The guinea-pig was clearly less sensitive than the rabbit or mouse. Owing to the relatively insoluble nature of zinc oxide and zinc undecylenate, dried residues of the compounds persisted on the skin surface for several hours after application. These increased with advancing hair regrowth. Hair regrowth was evident at all treated sites in the three species, but was possibly more advanced in those treated with zinc oxide, pyrithione or sulphate. Histological evidence of skin irritancy in animals treated with zinc chloride and to a lesser extent with zinc acetate included acanthosis, parakeratosis, hyperkeratosis and inflammatory changes in the epidermis and more superficial aspects of the dermis (Plate 1). Severe inflammatory changes with profound interfollicular acanthosis were frequently associated with acanthosis in distal aspects of follicular epithelia (Table 2). Follicular orifices were occasionally obstructed by granuioma and cell debris. Pilosebaceous follicles were also partly obstructed by residues of insoluble zinc compounds, but were rarely associated with inflammatory or hyperplastic changes in adjacent tissues. Hair growth and mitotic activity were not appreciably altered by the various treatments.
59
Occlusive patch tests in the rabbit As in the open patch tests, zinc chloride was the strongest irritant of the compounds tested. Erythema and ulceration were observed after 3 or 5 days. Zinc acetate was scored as a moderate irritant, whereas zinc sulphate, pyrithione and undecylenate were mild or insidious irritants. The level of erythema in the occlusive patch tests was marginally greater than in the open patches. Macroscopic observations of animals exposed to the zinc compounds for 3 days resembled those seen after 5 days. Hair regrowth was evident at treated and control sites for all zinc compounds, but was markedly less than in open tests. Histological changes seen in rabbit skin exposed to zinc compounds under occlusion resembled those seen in open patch tests, but tended to be more severe. Residues of zinc oxide and zinc undecylenate in the form of basophilic deposits were frequently present in follicular orifices. Obvious changes were not identified in the pilosebaceous apparatus. Hair papillae were normal.
Morin fluorescence for epidermal zinc deposition Zinc bound to epidermal keratin was demonstrated histologically in sections stained with morin dye and examined by ultraviolet light. In this study, blue-green fluorescence characteristic of zinc ion was present in all skin sections from rabbit, mouse and guinea-pig exposed to zinc chloride or zinc acetate and occasionally zinc sulphate (Table 3). In each case, the presence of zinc was demonstrated only in superficial layers of the epidermis and in follicular orifices (Plate 2). Fluorescence was prominent in regions of ulceration and parakeratosis.
Epidermal cell proliferation Epidermal cell proliferation under normal conditions is limited to the stratum germinativum of inter- and intrafollicular regions, and the hair papillae. In the mouse control group, the mitotic index 4 hr after the administration of vincristine was 8.59% (Table 4). Mice treated with zinc undecylenate and zinc sulphate, which induced negligible evidence of irritancy of epithelial hyperplasia, exhibited a similar mitotic index. However, zinc chloride and acetate, which were clearly irritant, induced epidermal hyperplasia and a greatly elevated mitotic index. Mice exposed to zinc oxide showed a slight increase in the epidermal mitotic index. In these animals, hair regrowth was more profound than in control animals.
Table I. Macroscopic observations in rabbit, mouse and guinea-pig skin exposed to zinc c o m p o u n d s for 5 days* Rabbit (open patch test) Zinc c o m p o u n d
No.
Oxide Chloride Sulphate Undecylenate Acetate Pyrithione 0 . 1 % Tween 80 Deionized water
4 4 4 4 4 4 4 4
* -
Irritancy + + + + + +
+ (4) (1) (1) (3) (2) -
Rabbit (occlusive patch test) No. 4 4 4 4 4 4 4 4
Mouse
Guinea-pig
Irritancy
No.
Irritancy
No.
Irritancy
Irritancy rating
-
6 6 6 6 6 6 6 6
+ + + (6) + (2) + (2) + + (6) +(I) -
8 8 8 8 8 8 8 8
+ + (3) + (1) -
Severe Low Low Moderate Low -
+ + + + + + +
+ (4) (1) (2) + (4) (2) -
N o obvious reaction; + slight irritancy--* ÷ + + severe irritancy; N o . o f animals showing responses in parentheses.
60
A . B . G . LANSDOWN
Table 2. Histological changes in the skin following application of zinc compounds in open patch tests* Zinc compound Rabbit Mouse Guinea-pig 20% Oxide 1 1 1 1% Chloride 5 5 4 I% Sulphate 2 1 1 20% Acetate 3 4 2 20% Pyrithione 3 3 1 20% Undecylenate 1 1 1 0.1% Tween 80 1 1 1 *1 = No evidence of damage; 2 ~ marginal epidermal thickening with no inflammatory change; 3 ffimild acanthosis with hyperkeratosis and some inflammatory change; 4=moderate acanthosis, hyperkeratosis, and inflammation with focal parakeratosis; 5 = profound epidermal hyperplasia with parakeratosis and ulceration.
Table
4. Influence of zinc compounds on epidermal cell proliferation in mouse skin
Zinc compound
Control 5 8.59 4- 1.90 Chloride 5 19.89 4- 2.82*** Acetate 5 28.15 4- 7.22*** Undecylenate 5 7.30 4- 0.64t Sulphate 5 8.96 4- 1.65t Pyrithione 5 10.26 4- 1.30t Oxide 5 10.74 _+ 1.36t • **Significantly different from control group (P < 0.001). tNS= Not significantly different from control group. Table 5. Microbiology in the skin following treatment with zinc compounds No. of skin samples showing infection*
Table 3. Morin fluorescence for zinc in skin* Morin fluorescence Zinc compound Oxide Chloride Sulphate Undecylenate Acetate Pyrithione
Rabbit +++ ++ -
Mouse +++ _ +++ -
Guinea-pig +++ + -
pH of zinc in solution/ suspension 7.4 5.6 5.7 5.9 6.0 6.6
* - No obvious reaction; 4- slight irritancy--*+ + + severe irritancy.
Microbiology T h e t h r e e species used in this s t u d y e x h i b i t e d similar p a t t e r n s o f skin flora, with Staphylococcus epidermidis a n d ~ - h a e m o l y t i c s t r e p t o c o c c i being pred o m i n a n t (Table 5). L i m i t e d e v i d e n c e is p r e s e n t e d h e r e to s h o w t h a t a n y o f these zinc c o m p o u n d s are bacteriostatic. Bacteria were n o t isolated f r o m areas o f m o u s e o r g u i n e a - p i g skin t r e a t e d with zinc p y r i t h i o n e , b u t g r o w t h o f S. epidermidis a n d c~h a e m o l y t i c s t r e p t o c o c c i was seen in areas o f r a b b i t skin t r e a t e d with this c o m p o u n d .
DISCUSSION Z i n c c o m p o u n d s like the a c e t a t e a n d chloride, w h i c h are a s t r i n g e n t in a q u e o u s solution, are clearly i r r i t a n t to t h e skin in the a n i m a l m o d e l s used in this study. O t h e r c o m p o u n d s like zinc oxide a n d zinc p y r i t h i o n e t h a t have a n e s t a b l i s h e d c o m mercial value, e x h i b i t a low p o t e n t i a l f o r skin irrit a t i o n a n d toxic c h a n g e even at h i g h levels o f exposure. A s s h o w n in p r e v i o u s studies, a s t r o n g c o r r e l a t i o n exists b e t w e e n t h e irritancy o f a x e n o b i o t i c s u b s t a n c e to the skin a n d its ability to b i n d with, a n d d e n a t u r e , t h e e p i d e r m a l k e r a t i n b a r r i e r ( L a n s d o w n , 1973 a n d 1974; L a n s d o w n a n d G r a s s o , 1972). I n t h e p r e s e n t
Epidermal mitotic index
No. of mice
Rabbit
(2) Zinc compound Oxide Chloride Sulphate Acetate Undecylenate Pyrithione Control
Guinea-pig
(2)
Mouse
(5)
S.c. ct-H.S.B.S.S.e, ct-H.S. Col. S.e. ~t-H.S. 1 2 1 -5 -1 --3 -2 I 2 4 2 l -1 2 --3 -2 --3 -1 1 -1 -2 -2 4 --
S.c. = Staphylococcus epidermidis ~-H.s. = ~-haemolytic streptococci; B.s. = Bacillus sp.; Col. = Miscellaneous coliforms; *No of skin samples in parentheses. w o r k , t h e c o m p a r a t i v e k e r a t i n b i n d i n g ability o f t h e zinc ion f r o m t h e six c o m p o u n d s is d e m o n s t r a t e d b y m o r i n fluorescence. A l t h o u g h several o f the s o l u t i o n s / s u s p e n s i o n s t h a t were a p p l i e d to t h e skin surface were m o d e r a t e l y acidic, it is unlikely t h a t t h e p H per se w a s a n i m p o r t a n t c o n t r i b u t o r y f a c t o r in the irritancy seen ( L a n s d o w n , 1973). H o w e v e r , it c o u l d c o n c e i v a b l y p r o v i d e a n e n v i r o n m e n t a l m e d i u m fav o u r i n g the i n t e r a c t i o n o f free zinc i o n a n d e p i d e r m a l keratin. Earlier r e p o r t s have a l l u d e d to the antibacterial p r o p e r t i e s o f c e r t a i n zinc c o m p o u n d s ( I m o k a w a et al., 1982; S n y d e r et al., 1977). This has n o t b e e n e s t a b l i s h e d in t h e p r e s e n t study, w h e r e n o n e o f t h e six c o m p o u n d s tested p r o v e d c o n s i s t e n t l y effective a g a i n s t c o m m o n strains o f b a c t e r i a in t h e t h r e e species o f test animal. Zinc p y r i t h i o n e , w h i c h was p o s s i b l y b a c t e r i o s t a t i c in t w o species, h a s previously been s h o w n to be ineffective a g a i n s t G r a m positive o r g a n i s m s in h u m a n trials ( M a r k s et al., 1985). Zinc is a n a c k n o w l e d g e d c o n s t i t u e n t o f at least 40 e n z y m e s a n d is essential for n o r m a l cell g r o w t h , b i o s y n t h e s i s a n d differentiation (Keller a n d M a n n , 1940; K i r c h e s s n e r et al., 1976; L i n d e m a n a n d Mills, 1980). H o w e v e r , a l t h o u g h high zinc c o n c e n t r a t i o n s
Plate 1. Rabbit skin exposed to 1% zinc chloride (0.5 ml) daily in open patch tests for 5 days, showing pronounced epidermial thickening, superficial ulceration and moderate dermal inflammatory cell infiltration. Haematoxylin and eosin, x 76. Plate 2. Granules of zinc oxide deposited (arrows) in the orifices of hair follicles in rabbit skin treated with 20% zinc oxide suspension daily for 5 days. Note lack of epidermal hyperplasia and dermal inflammatory changes. Haematoxylin and eosin, x 125. Plate 3. Zinc deposits in epidermal keratin in the hyperkeratotic epidermis in the region o f a hair follicle from a TO mouse treated with 1.0% zinc chloride daily for 5 days. Morin fluorescence x 75.
r~
t~
63
Interspecies responses to zinc compounds have been shown to stimulate blastogenic transformation in cultured human peripheral lymphocytes (Ruhl et al., 1971), there seems to be no good evidence that high zinc concentrations are conducive of increased proliferation of these or other cells in vivo. Previous studies have demonstrated that zinc concentrates in the region of skin wounds (Savlov et al., 1962), but that this is not associated with increased tensile strength in the regenerated tissue (Groundwater and MacLeod, 1970; O'Riain et al., 1968). It seems that if zinc does exert a beneficial influence on wound healing, it does so at the reepithelialization stage (Cohen, 1968). The present experiments have demonstrated that compounds like zinc oxide, zinc pyrithione and possibly zinc sulphate are capable of inducing a mild but not statistically significant epidermal hyperplasia in normal skin following topical application, and at concentrations that proved to be non-irritant. On the other hand, the severe acanthosis and hyperkeratosis seen in response to zinc chloride or acetate must be regarded as a regenerative response, namely, a reconstitution of epidermis lost or damaged by the corrosive influence of the solution applied. In other in vivo studies where 1% zinc pyrithione was applied in a shampoo to rat skin for 4 consecutive days, DNA synthesis was marginally depressed (Gibson, et al., 1985). This observation, which is at variance with the results of the present study, may be explained in part by differences in the experimental model and the sensitivity of the analyses used. Further studies are required. Alternatively, it may be that the increased mitotic activity with marginal to moderate changes in epidermal thickness reflect a subtle toxic change that does not give risk to frank irritancy with inflammatory changes. If the epidermal hyperplasia, albeit at a low level, is attributable to a mitogenic rather than to a toxic effect, then the implications in terms of wound healing are considerable. The present results do not show the amount of zinc ion passing percutaneously, but it is likely to be low (Yankell, 1969). However, as zinc is a trace metal it is conceivable that even a small increase in local concentration will enhance the mitotic activity in the interfollicular epidermis or hair papillae. Alopecia and impaired hair growth are characteristic of zinc deficiency in humans and animals (Ecker and Schroeter, 1978; Moynahan, 1974; Prasad, 1966; Tucker et al., 1976). The zinc concentrations in the hair may serve as an accurate guide to whole body levels (Strain et al., 1966). Although the present experiments do not provide information on hair zinc concentrations after topical treatments, they do give provocative evidence that supplementary zinc absorbed percutaneously can enhance the regrowth of hair in shaved or depilated areas. Further work is necessary in this field. In conclusion, it seems likely that the responses in the skin reflect a balance between the physiological and toxic effects of the compound applied. They depend on the ability of the xenobiotic to react with and denature the epidermal keratin, the extent to which the zinc ion concentrates in the skin, and the capacity of the tissue to respond or tolerate the zinc overload.
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Molokhia M. H. and Portnoy B. (1969) Neutron activation analysis of trace elements in skin. III. Zinc in normal skin. British Journal of Dermatology $1, 759-762. Moynahan E. J. (1974) Acrodermatitis enteropathica, a lethal inherited human zinc deficiency disorder. Lancet i, 399-400. Ohlen B. and Scott D. W. (1986) Zinc-responsive dermatitis in puppies. Canine Pracitce 13, 6-10. O'Riain S., Copenhagen H. J. and Calnan J. S. (1968) The effect of zinc sulphate on the healing of incised wounds in rats. British Journal of Plastic Surgery 21, 240-243. Pories W. J., Henzel J. H., Rob C. G. and Strain W. H. (1967) Acceleration of healing with zinc sulphate. Annals of Surgery 165, 432-436. Prasad A. S. (1966) Metabolism of zinc and its deficiency in the human subject. In Zinc Metabolism. Edited by A. S. Prasad. p. 250. C. C. Thomas, Springfield, IL. Raulin J. (1869) Etudes cliniques sur la vegetation. Annales des Sciences Naturelles Botanique et Biologie Vegetale 11, 93. Ruhl H., Kirchner H. and Bochert G. (1971) Kinetics of the Zn 2 stimulation of human peripheral lymphocytes. Pro-
ceedings of the Society for Experimental Biology and Medicine 137, 1089-1092. Savlov E. D., Strain W. H. and Huegin F. (1962) Radiozinc studies in experimental wound healing. Journal of Surgical Research 2, 209-212. Schroeder H. A., Nason A. P., Tipton I. H. and Balassa J. J. (1967) Essential trace elements in man: zinc relative to
environmental cadmium. Journal of Chronic Diseases 20, 179-210. Skog E. and Wahlberg J. E. (1964) A comparative investigation of the percutaneous absorption of metal compounds in the guinea pig by means of the radioactive isotopes 51Cr, SSCo, eSZn, U°Ag, ttSCd, 2°3Hg. Journal of Investigatwe Dermatology 43, 187-192. Snyder D. R., Gralla E. J., Coleman G. L. and Wedig J. H. (1977) Preliminary neurological evaluation of generalized weakness in zinc pyrithione-treated rats. Food and Cosmetics Toxicology 15, 43-47. Strain W. H., Steadman L. T., Lankau C. A., Berliner W. P. and Pories W. J. (1966) Analysis of zinc levels for the diagnosis of zinc deficiency in man. Journal of Laboratory and Clinical Medicine 68, 244-249. Tucker H. F. and Salmon D. W. (1955) Parakeratosis in zinc deficient disease in the pig. Proceedings of the Society for Experimental Biology and Medicine 88, 613-616. Tucker S. B., Schroeter A. L., Brown P. W. and McCall J. T. (1976) Acquired zinc deficiency, cutaneous manifestations typical of acrodermatitis enteropathica. Journal of the American Medical Association 235, 2399-2402. Van Herck H., Van Wouwe J. P., Veldhuizen M., Baumanns V., Stafleu F. R. and Beynon A. C. (1989) Clinical examination of weanling rats and early zinc deficiency. Laboratory Animals 23, 328-332. Yankell S. L. (1969) Translocation of topically applied zinc. In Pharmacology and the Skin. Edited by W. Montagua, R. B. Stoughton and E. Van Scott. Advances in the Biology of the Skin XII. 12, 516-517.
0278-6915/91 $3.00+ 0.00 Copyright© 1991PergamonPressple
INTERSPECIES VARIATIONS IN RESPONSE TO TOPICAL APPLICATION OF SELECTED ZINC COMPOUNDS A. B. G. LANSDOWN Chafing Cross and Westminster Medical School, Department of Comparative Biology, London W6 8RP, UK (Received 27 June 1990; revisions received 19 September 1990)
Abstract--The dermal irritancy of six zinc compounds was examined in three animal models. In open patch tests involving five daily applications, zinc chloride (1% aqueous solution) was severely irritant in rabbit, guinea-pig and mouse tests, inducing epidermal hyperplasia and ulceration; aqueous zinc acetate (20%) was slightly less irritant. Zinc oxide (20% suspension dilute Tween 80), zinc sulphate (1% aqueous solution) and zinc pyrithione (20% suspension) were not overtly irritant, but induced a marginal epidermal hyperplasia and increased hair growth. Zinc undeeylenate (20% suspension) was not irritant. Epidermal irritancy in these studies is related to the interaction of zinc ion with epidermal keratin. The compounds studied were not consistently baeteriostatic in the three species tested.
INTRODUCTION
the action of zinc in epidermal repair following incisional lesion, varicose ulcer or other forms of benign injury. Considerable clinical interest and commercial value has been attached to the premise that zinc accelerates the healing of skin wounds (Hallbook and Lanner, 1972; Hallmans, 1978; Henzel et al., 1970; Hussain, 1969; Pories et al., 1967), but the subject is controversial and contradictory evidence is available (Barcia, 1970; Groundwater and MacLeod, 1970; Lancet, 1975). Clearly, if zinc is beneficial to wound healing in the skin, one would expect it to promote cell proliferation, migration and differentiation in regenerating tissues. This effect is still not established clinically or experimentally (Marks et al., 1985). A variety of preparations containing zinc is available to treat skin lesions, but in many cases their action on the epidermis is unclear. As a prelude to further studies to define more clearly the potential value of zinc in wound healing, the study presented here was designed to observe the influence of specific zinc compounds on the epidermis of three animal models commonly used in experimental dermatology. This study resembles an earlier investigation that compared the irritancy of several aluminium compounds (Lansdown, 1973 and 1974). Like aluminium, zinc salts of strong acids are astringent and corrosive to the skin at high concentrations. However, whereas aluminium has no obvious role in skin physiology, zinc is an essential trace metal. Therefore, it is of additional interest to examine the extent to which exaggerated levels of topical zinc enhance the physiological action of the element, or induce toxic changes.
Zinc is an essential nutrient and constituent of all biological systems (Raulin, 1869). After iron, it is the most abundant trace metal in the mammalian body. Although much is still unclear about the physiological role of zinc, it is now known to be a component of at least 40 enzyme systems, including alkaline phosphatase, carbonic anhydrase, dehydrogenases, carboxypeptidases and D N A / R N A polymerases (Keller and Mann, 1940; Kirchessner et al., 1976). Zinc concentrations tend to be higher in the skin, red blood cells and tissues that exhibit high proliferative activity (Schroeder et aL, 1967). In human skin, six times more zinc is found in the epidermis than in the dermis (Molokhia and Portnoy, 1969). The importance of zinc in the growth and maintenance of the functional integrity of the skin is unequivocal. The rare lethal condition acrodermatitis enteropathica, which results from a hereditary hypozincaemia, is well documented as are skin lesions induced experimentally in laboratory animals fed zinc-deficient diets (DeGryse et al., 1987; Ecker and Schroeter, 1978; Moynahan, 1974; Ohlen and Scott, 1986; Prasad, 1966; Strain et al., 1966; Tucker et al., 1976). Zinc-responsive dermatosis occurs naturally in certain strains of dog (Tucker and Salmon, 1955; Van Herck et al., 1989). These conditions show similar dermatological features namely hyperkeratosis, parakeratosis, hypopigmentation and alopecia. This suggests that despite obvious morphological differences in the skin between species, animal models may be used to provide beneficial information that can advance our understanding of the pathophysiological role of zinc in human skin. Zinc concentrations are normally maintained in the body by intestinal absorption, but experimental evidence has shown that low levels of zinc may be absorbed percutaneously in zinc-deficient animals to restore norrnozincaemia (Kapur et al., 1974; Keen and Hurley, 1977; Skog and Wahlberg, 1964; Yankell, 1969). This may have further implications in
MATERIALS AND METHODS
Animals Mice. Animals used were TO (outbred) and AG2 (inbred) strains bred under barrier-maintained specified pathogen-free conditions at the Chafing Cross and Westminster Medical School. Male mice
57
58
A.B.G. L^NSDOWS
were 8-10 wk old, and housed in a conventional environment (12-hr light/dark cycle, 2 2 _ 2°C and 55% relative humidity) and fed a complete rodent diet (CRM, Biosure, Cambridge, UK) (zinc content 65 mg/kg, w/w) and deionized water ad lib. Rabbits. Animals were New Zealand white males weighing 2.5--4.0kg, which were reared at the Chafing Cross and Westminster Medical School. They were housed singly under conditions of a 12-hr light/dark cycle, at 18 _ 2°C and 45% relative humidity. Their diet consisted of R14 pellets (Biosure) (zinc content 74 mg/kg, w/w) and tap-water. Guinea-pigs. Male animals of the Dunkin-Hartley white strain (250-400 g) were supplied by HarlanPorcellus (Heathfield, Sussex, UK). They were fed an RGP diet (Biosure) (zinc content 65 mg/kg, w/w) and maintained in an environment similar to that of the mice.
Chemicals The zinc compounds were supplied by the Sigma Chemical Co. Ltd (Poole, Dorset, UK) and had a minimum purity of 98%. The concentrations used were those that had been shown to be non-irritant in preliminary acute studies in white (TO) mice (mice back skin was treated with 0.5 ml of the test compound at 0.1, 1.0, 10 or 20% and examined for erythema after 24 hr). The following preparations were applied to the animal skins: (i) 1% (w/v) zinc chloride solution in deionized water; (ii) 1% (w/v) zinc sulphate solution in deionized water; (iii) 20% (w/v) zinc acetate solution in deionized water; (iv) 20% (w/v) zinc oxide suspension in 0.1% Tween 80; (v) 20% (w/v) zinc undecylenate suspension in 0.1% Tween 80; and (vi) 20% (w/v) zinc pyrithione suspension in 0.1% Tween 80. Control areas of skin were treated with either deionized water or Tween 80, both of which are non-irritant in subacute skin tests in mice (Lansdown and Grasso, 1972). In each case, test solutions were prepared 24 hr before use and stored at 4°C. The pH of each preparation was measured immediately before application (Table 3). Experimental procedures Four experiments were set up to compare the irritancy of the six zinc compounds. These included two studies in rabbits (one under open patch test conditions, and one using occlusive conditions), and one each in guinea-pigs and mice. In each case, test sites (5 cm 2) were prepared in the mid-dorsal regions. The hair coat was clipped closely and the skin cleansed with 70% alcohol and allowed to dry before treatment with zinc compounds. In the initial rabbit study, 4 animals were acclimatized to conventional restraining boxes. Eight test sites were designated, four on each side of the mid-dorsal line. Zinc compounds (or controls) were applied daily (0.5 ml) at 09.00-09.30 hr on 5 successive days. The animals were restrained for 30 min to allow test sites to dry. In a second rabbit experiment, 4 rabbits were prepared as above, but the 0.5 ml of test compound was applied to the skin on a sterile gauze pad that was secured by a hypoallergenic adhesive tape (Draize
et al., 1944; Lansdown, 1978). The trunks of these animals were wrapped in rubberized fabric and impermeable dressing for 3 days. At this stage, dressings were removed and skin sites evaluated for irritancy. Two rabbits were killed at this time by using excess barbiturate anaesthetic. The remaining animals were redressed with freshly impregnated gauze pads and then re-examined and killed after a further 2 days. In the guinea-pig experiment, 8 animals were prepared by shaving the dorsal skin to provide three treatment sites (5 cm:). A total of three treatment sites was thus available for each of the test and control solutions/suspensions. The guinea-pigs were treated daily (09.00-09.30 hr) for 5 consecutive days. The animals were restrained until the test sites had dried. Open patch tests were conducted in mice in eight groups of 6 animals. Sites of 5 cm 2 were treated with 0.5 ml of the test or control agent for 5 consecutive days. Animals were gently anaesthetized with ether until the test sites had dried. Twenty-four hr after the fifth daily treatment, rabbits, guinea-pigs and mice were killed using excess barbiturate anaesthetic or cervical dislocation (mice). Representative samples of each test and control skin site were preserved in 10% phosphate buffered formalin for histology. Thin sections cut along the anterior-posterior axis were stained with haematoxylin and eosin, or with morin dye, which fluoresces blue-green in the presence of zinc ions and ultraviolet light. Epidermal cell kinetics The influence of the various zinc preparations on the mitotic behaviour of the dorsal skin epidermis was examined. The experiment was set up identically to the mouse experiment, except that groups of 5 A2G mice were used for each treatment regimen. Twenty-four hr after the final treatment, each mouse received a single ip injection of 0.1 mg vincristine (Oncovin, Lilly, Basingstoke, Hants., UK) in 0.25 ml of normal saline (Bullough and Laurence, 1966). The animals were killed by cervical dislocation after 4 hr and three strips of skin from the treatment sites were preserved in 10% formalin for histological examination. Sections were stained with haematoxylin and eosin, and arrested mitoses per 1000 cells were counted. The mitotic index was calculated as: total no. of arrested mitoses x 100 total no. of cells counted Where the mouse skins exhibited acanthotic changes, and where mitoses occurred above the stratum germinativum, mitotic counts only included cells in the striatum germinativum and in the first layer of the stratum spinosum. The mitotic index was calculated for each mouse. Microbiology In view of the observation that certain zinc compounds exhibit antibiotic properties (Imokawa et al., 1982; Snyder et al., 1977), fresh skin samples were obtained from the rabbits, guinea-pigs and mice at the end of the experimental period. They were routinely smeared across sterile blood agar plates, incubated at 37°C, and examined for bacterial
Interspecies responses to zinc compounds growths 24 hr later using standard criteria. Previous quality assurance studies have shown an absence of ectoparasites, mycoplasma and viral infections in animals bred and maintained in the animal facility of the Charing Cross and Westminster Medical School.
Statistics Where appropriate, statistical comparisons were made with Student's t-test. RESULTS
Open patch tests Zinc chloride was clearly the strongest irritant of the compounds tested in open patch tests in the three species (Table 1). From the first application, skin test sites became erythematous, and by the fifth day they showed ulcerative changes. Lower levels of erythema were seen following treatment with zinc acetate, sulphate, pyrithione or undecylenate. These compounds did not induce ulceration or scaling in the test period. Zinc oxide suspension, dilute Tween 80 and deionized water also did not show an obvious effect. The guinea-pig was clearly less sensitive than the rabbit or mouse. Owing to the relatively insoluble nature of zinc oxide and zinc undecylenate, dried residues of the compounds persisted on the skin surface for several hours after application. These increased with advancing hair regrowth. Hair regrowth was evident at all treated sites in the three species, but was possibly more advanced in those treated with zinc oxide, pyrithione or sulphate. Histological evidence of skin irritancy in animals treated with zinc chloride and to a lesser extent with zinc acetate included acanthosis, parakeratosis, hyperkeratosis and inflammatory changes in the epidermis and more superficial aspects of the dermis (Plate 1). Severe inflammatory changes with profound interfollicular acanthosis were frequently associated with acanthosis in distal aspects of follicular epithelia (Table 2). Follicular orifices were occasionally obstructed by granuioma and cell debris. Pilosebaceous follicles were also partly obstructed by residues of insoluble zinc compounds, but were rarely associated with inflammatory or hyperplastic changes in adjacent tissues. Hair growth and mitotic activity were not appreciably altered by the various treatments.
59
Occlusive patch tests in the rabbit As in the open patch tests, zinc chloride was the strongest irritant of the compounds tested. Erythema and ulceration were observed after 3 or 5 days. Zinc acetate was scored as a moderate irritant, whereas zinc sulphate, pyrithione and undecylenate were mild or insidious irritants. The level of erythema in the occlusive patch tests was marginally greater than in the open patches. Macroscopic observations of animals exposed to the zinc compounds for 3 days resembled those seen after 5 days. Hair regrowth was evident at treated and control sites for all zinc compounds, but was markedly less than in open tests. Histological changes seen in rabbit skin exposed to zinc compounds under occlusion resembled those seen in open patch tests, but tended to be more severe. Residues of zinc oxide and zinc undecylenate in the form of basophilic deposits were frequently present in follicular orifices. Obvious changes were not identified in the pilosebaceous apparatus. Hair papillae were normal.
Morin fluorescence for epidermal zinc deposition Zinc bound to epidermal keratin was demonstrated histologically in sections stained with morin dye and examined by ultraviolet light. In this study, blue-green fluorescence characteristic of zinc ion was present in all skin sections from rabbit, mouse and guinea-pig exposed to zinc chloride or zinc acetate and occasionally zinc sulphate (Table 3). In each case, the presence of zinc was demonstrated only in superficial layers of the epidermis and in follicular orifices (Plate 2). Fluorescence was prominent in regions of ulceration and parakeratosis.
Epidermal cell proliferation Epidermal cell proliferation under normal conditions is limited to the stratum germinativum of inter- and intrafollicular regions, and the hair papillae. In the mouse control group, the mitotic index 4 hr after the administration of vincristine was 8.59% (Table 4). Mice treated with zinc undecylenate and zinc sulphate, which induced negligible evidence of irritancy of epithelial hyperplasia, exhibited a similar mitotic index. However, zinc chloride and acetate, which were clearly irritant, induced epidermal hyperplasia and a greatly elevated mitotic index. Mice exposed to zinc oxide showed a slight increase in the epidermal mitotic index. In these animals, hair regrowth was more profound than in control animals.
Table I. Macroscopic observations in rabbit, mouse and guinea-pig skin exposed to zinc c o m p o u n d s for 5 days* Rabbit (open patch test) Zinc c o m p o u n d
No.
Oxide Chloride Sulphate Undecylenate Acetate Pyrithione 0 . 1 % Tween 80 Deionized water
4 4 4 4 4 4 4 4
* -
Irritancy + + + + + +
+ (4) (1) (1) (3) (2) -
Rabbit (occlusive patch test) No. 4 4 4 4 4 4 4 4
Mouse
Guinea-pig
Irritancy
No.
Irritancy
No.
Irritancy
Irritancy rating
-
6 6 6 6 6 6 6 6
+ + + (6) + (2) + (2) + + (6) +(I) -
8 8 8 8 8 8 8 8
+ + (3) + (1) -
Severe Low Low Moderate Low -
+ + + + + + +
+ (4) (1) (2) + (4) (2) -
N o obvious reaction; + slight irritancy--* ÷ + + severe irritancy; N o . o f animals showing responses in parentheses.
60
A . B . G . LANSDOWN
Table 2. Histological changes in the skin following application of zinc compounds in open patch tests* Zinc compound Rabbit Mouse Guinea-pig 20% Oxide 1 1 1 1% Chloride 5 5 4 I% Sulphate 2 1 1 20% Acetate 3 4 2 20% Pyrithione 3 3 1 20% Undecylenate 1 1 1 0.1% Tween 80 1 1 1 *1 = No evidence of damage; 2 ~ marginal epidermal thickening with no inflammatory change; 3 ffimild acanthosis with hyperkeratosis and some inflammatory change; 4=moderate acanthosis, hyperkeratosis, and inflammation with focal parakeratosis; 5 = profound epidermal hyperplasia with parakeratosis and ulceration.
Table
4. Influence of zinc compounds on epidermal cell proliferation in mouse skin
Zinc compound
Control 5 8.59 4- 1.90 Chloride 5 19.89 4- 2.82*** Acetate 5 28.15 4- 7.22*** Undecylenate 5 7.30 4- 0.64t Sulphate 5 8.96 4- 1.65t Pyrithione 5 10.26 4- 1.30t Oxide 5 10.74 _+ 1.36t • **Significantly different from control group (P < 0.001). tNS= Not significantly different from control group. Table 5. Microbiology in the skin following treatment with zinc compounds No. of skin samples showing infection*
Table 3. Morin fluorescence for zinc in skin* Morin fluorescence Zinc compound Oxide Chloride Sulphate Undecylenate Acetate Pyrithione
Rabbit +++ ++ -
Mouse +++ _ +++ -
Guinea-pig +++ + -
pH of zinc in solution/ suspension 7.4 5.6 5.7 5.9 6.0 6.6
* - No obvious reaction; 4- slight irritancy--*+ + + severe irritancy.
Microbiology T h e t h r e e species used in this s t u d y e x h i b i t e d similar p a t t e r n s o f skin flora, with Staphylococcus epidermidis a n d ~ - h a e m o l y t i c s t r e p t o c o c c i being pred o m i n a n t (Table 5). L i m i t e d e v i d e n c e is p r e s e n t e d h e r e to s h o w t h a t a n y o f these zinc c o m p o u n d s are bacteriostatic. Bacteria were n o t isolated f r o m areas o f m o u s e o r g u i n e a - p i g skin t r e a t e d with zinc p y r i t h i o n e , b u t g r o w t h o f S. epidermidis a n d c~h a e m o l y t i c s t r e p t o c o c c i was seen in areas o f r a b b i t skin t r e a t e d with this c o m p o u n d .
DISCUSSION Z i n c c o m p o u n d s like the a c e t a t e a n d chloride, w h i c h are a s t r i n g e n t in a q u e o u s solution, are clearly i r r i t a n t to t h e skin in the a n i m a l m o d e l s used in this study. O t h e r c o m p o u n d s like zinc oxide a n d zinc p y r i t h i o n e t h a t have a n e s t a b l i s h e d c o m mercial value, e x h i b i t a low p o t e n t i a l f o r skin irrit a t i o n a n d toxic c h a n g e even at h i g h levels o f exposure. A s s h o w n in p r e v i o u s studies, a s t r o n g c o r r e l a t i o n exists b e t w e e n t h e irritancy o f a x e n o b i o t i c s u b s t a n c e to the skin a n d its ability to b i n d with, a n d d e n a t u r e , t h e e p i d e r m a l k e r a t i n b a r r i e r ( L a n s d o w n , 1973 a n d 1974; L a n s d o w n a n d G r a s s o , 1972). I n t h e p r e s e n t
Epidermal mitotic index
No. of mice
Rabbit
(2) Zinc compound Oxide Chloride Sulphate Acetate Undecylenate Pyrithione Control
Guinea-pig
(2)
Mouse
(5)
S.c. ct-H.S.B.S.S.e, ct-H.S. Col. S.e. ~t-H.S. 1 2 1 -5 -1 --3 -2 I 2 4 2 l -1 2 --3 -2 --3 -1 1 -1 -2 -2 4 --
S.c. = Staphylococcus epidermidis ~-H.s. = ~-haemolytic streptococci; B.s. = Bacillus sp.; Col. = Miscellaneous coliforms; *No of skin samples in parentheses. w o r k , t h e c o m p a r a t i v e k e r a t i n b i n d i n g ability o f t h e zinc ion f r o m t h e six c o m p o u n d s is d e m o n s t r a t e d b y m o r i n fluorescence. A l t h o u g h several o f the s o l u t i o n s / s u s p e n s i o n s t h a t were a p p l i e d to t h e skin surface were m o d e r a t e l y acidic, it is unlikely t h a t t h e p H per se w a s a n i m p o r t a n t c o n t r i b u t o r y f a c t o r in the irritancy seen ( L a n s d o w n , 1973). H o w e v e r , it c o u l d c o n c e i v a b l y p r o v i d e a n e n v i r o n m e n t a l m e d i u m fav o u r i n g the i n t e r a c t i o n o f free zinc i o n a n d e p i d e r m a l keratin. Earlier r e p o r t s have a l l u d e d to the antibacterial p r o p e r t i e s o f c e r t a i n zinc c o m p o u n d s ( I m o k a w a et al., 1982; S n y d e r et al., 1977). This has n o t b e e n e s t a b l i s h e d in t h e p r e s e n t study, w h e r e n o n e o f t h e six c o m p o u n d s tested p r o v e d c o n s i s t e n t l y effective a g a i n s t c o m m o n strains o f b a c t e r i a in t h e t h r e e species o f test animal. Zinc p y r i t h i o n e , w h i c h was p o s s i b l y b a c t e r i o s t a t i c in t w o species, h a s previously been s h o w n to be ineffective a g a i n s t G r a m positive o r g a n i s m s in h u m a n trials ( M a r k s et al., 1985). Zinc is a n a c k n o w l e d g e d c o n s t i t u e n t o f at least 40 e n z y m e s a n d is essential for n o r m a l cell g r o w t h , b i o s y n t h e s i s a n d differentiation (Keller a n d M a n n , 1940; K i r c h e s s n e r et al., 1976; L i n d e m a n a n d Mills, 1980). H o w e v e r , a l t h o u g h high zinc c o n c e n t r a t i o n s
Plate 1. Rabbit skin exposed to 1% zinc chloride (0.5 ml) daily in open patch tests for 5 days, showing pronounced epidermial thickening, superficial ulceration and moderate dermal inflammatory cell infiltration. Haematoxylin and eosin, x 76. Plate 2. Granules of zinc oxide deposited (arrows) in the orifices of hair follicles in rabbit skin treated with 20% zinc oxide suspension daily for 5 days. Note lack of epidermal hyperplasia and dermal inflammatory changes. Haematoxylin and eosin, x 125. Plate 3. Zinc deposits in epidermal keratin in the hyperkeratotic epidermis in the region o f a hair follicle from a TO mouse treated with 1.0% zinc chloride daily for 5 days. Morin fluorescence x 75.
r~
t~
63
Interspecies responses to zinc compounds have been shown to stimulate blastogenic transformation in cultured human peripheral lymphocytes (Ruhl et al., 1971), there seems to be no good evidence that high zinc concentrations are conducive of increased proliferation of these or other cells in vivo. Previous studies have demonstrated that zinc concentrates in the region of skin wounds (Savlov et al., 1962), but that this is not associated with increased tensile strength in the regenerated tissue (Groundwater and MacLeod, 1970; O'Riain et al., 1968). It seems that if zinc does exert a beneficial influence on wound healing, it does so at the reepithelialization stage (Cohen, 1968). The present experiments have demonstrated that compounds like zinc oxide, zinc pyrithione and possibly zinc sulphate are capable of inducing a mild but not statistically significant epidermal hyperplasia in normal skin following topical application, and at concentrations that proved to be non-irritant. On the other hand, the severe acanthosis and hyperkeratosis seen in response to zinc chloride or acetate must be regarded as a regenerative response, namely, a reconstitution of epidermis lost or damaged by the corrosive influence of the solution applied. In other in vivo studies where 1% zinc pyrithione was applied in a shampoo to rat skin for 4 consecutive days, DNA synthesis was marginally depressed (Gibson, et al., 1985). This observation, which is at variance with the results of the present study, may be explained in part by differences in the experimental model and the sensitivity of the analyses used. Further studies are required. Alternatively, it may be that the increased mitotic activity with marginal to moderate changes in epidermal thickness reflect a subtle toxic change that does not give risk to frank irritancy with inflammatory changes. If the epidermal hyperplasia, albeit at a low level, is attributable to a mitogenic rather than to a toxic effect, then the implications in terms of wound healing are considerable. The present results do not show the amount of zinc ion passing percutaneously, but it is likely to be low (Yankell, 1969). However, as zinc is a trace metal it is conceivable that even a small increase in local concentration will enhance the mitotic activity in the interfollicular epidermis or hair papillae. Alopecia and impaired hair growth are characteristic of zinc deficiency in humans and animals (Ecker and Schroeter, 1978; Moynahan, 1974; Prasad, 1966; Tucker et al., 1976). The zinc concentrations in the hair may serve as an accurate guide to whole body levels (Strain et al., 1966). Although the present experiments do not provide information on hair zinc concentrations after topical treatments, they do give provocative evidence that supplementary zinc absorbed percutaneously can enhance the regrowth of hair in shaved or depilated areas. Further work is necessary in this field. In conclusion, it seems likely that the responses in the skin reflect a balance between the physiological and toxic effects of the compound applied. They depend on the ability of the xenobiotic to react with and denature the epidermal keratin, the extent to which the zinc ion concentrates in the skin, and the capacity of the tissue to respond or tolerate the zinc overload.
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